Reverse transcription (RT) and the polymerase chain reaction (PCR) are critical to many molecular biology and related applications, particularly gene expression analysis applications. In these applications, reverse transcription is used to prepare template DNA from an initial RNA sample, e.g. mRNA, which template DNA is then amplified using PCR to produce a sufficient amount of amplified product for the application of interest. The RT and PCR steps of DNA amplification can be carried out as a two step or one step process.
In two step processes, the first step involves synthesis of first strand cDNA with a reverse transcriptase, e.g. MMLV-RT, following by a second PCR step. In certain protocols, these steps are carried out in separate reaction tubes. In these two tube protocols, following reverse transcription of the initial RNA template in the first tube, an aliquot of the resultant product is then placed into the second PCR tube and subjected to PCR amplification.
In a second type of two-step process, both RT and PCR are carried out in the same tube using a compatible RT and PCR buffer. In certain embodiments of single tube protocols, reverse transcription is carried out first, followed by addition of PCR reagents to the reaction tube and subsequent PCR.
In an effort to further expedite and simplify RT-PCR procedures, a variety of one step RT-PCR protocols have been developed. See e.g. the Relevant Literature section, supra. However, there is still room for improvement of these methods in a number of areas, including sensitivity, efficiency, and the like.
Accordingly, there is continued interest in the development of additional one step RT-PCR protocols, where a highly efficient and sensitive protocol is of particular interest.
Relevant Literature
See Blain & Goff, J. Biol. Chem. (1993) 5: 23585-23592; Blain & Goff, J. Virol. (1995) 69:4440-4452; Sellner et al., J. Virol. Method. (1994) 49:47-58; PCR, ESSENTIAL TECHNIQUES (ed. J. F. Burke, J. Wiley & Sons, New York)(1996) pp61-63; 80-81; SuperScript One-Step RT-PCR System description on the world-wide web at http://www.lifetech.com/world_whatsnew/archive/nz.sub.-- 1.sub.-- 3.html; Access RT-PCR System and Access RT-PCR Introductory System described on the world wide web at http://www.promega.com/tbs/tb220/tb220.html; and AdvanTaq & AdvanTaq Plus PCR kits and User Manual available at www.clontech.com at least as early as Sep. 15, 1999.